The effects of low dose (less than 1 Rad) X-rays on the erythropoietic marrow

Adult female Sprague-Dawley rats were exposed (200 kvp X-rays) to whole body doses of 22-1320 mrad and examined for changes in the level of red blood cell precursors (RBCp) in the marrow at 5–30 weeks post-irradiation, under nonbled and phlebotomy-induced anemic stress conditions. Increases in the RBCp %, RBCp/mg marrow, and RBCp/skeleton under nonbled conditions, and a suppressed erythroid response to an induced anemia, were found after acute doses in the range of at least 70 mrad. Dosages of 22 or 44 mrad that induced no measurable changes when applied only once were found to be effective when they were employed 4 or 2 times/week, respectively. The results suggested the presence of a linear-quadratic dose-response relationship in which the quadratic function exists between 88 and 981 mrad, and the linear dependency, below 88 mrad.     

Influence of DNA repair defects (rad1, rad52) on nitrogen mustard mutagenesis in yeast.

Summary Nitrogen mustard (HN2) mutagenesis of a plasmid-borne copy of the Saccharomyces cerevisiae SUP4-o gene was examined in a repair-proficient yeast strain and isogenic derivatives defective for excision (radl) or DNA double-strand break (rad52) repair. The excision repair deficiency sensitized the cells to killing by HN2 and abolished mutation induction. Inactivation of RAD52 had no influence on the lethality of HN2 treatment but diminished the induced mutation frequency by 50% at all doses tested. DNA sequence analysis of HN2-induced SUP4-o mutations suggested that RAD52 contributed to the production of basepair substitutions at G·C sites. The rad52 defect appeared to alter the distribution of G·C A·T transitions in SUP4-o relative to the distribution for the wild-type strain. This difference did not seem to be due to an effect of RAD52 on the relative fractions of HN2-induced transitions at localized (flanked by A·T pairs) or contiguous (flanked by at least one G·C pair) G·C sites but instead to an influence on the strand specificity of HN2 mutagenesis. In the repair-proficient strain, the transitions showed a small bias for sites having the guanine on the transcribed strand and this preference was eliminated by inactivation of RAD52.     

Human lymphocytes exposed to low doses of X-rays are less susceptible to radiation-induced mutagenesis

Human lymphocytes exposed to low doses of X-rays become refractory to the subsequent induction of chromosomal damage by high doses of radiation (Shadley and Wolff, 1987). The current study was designed to test the effect of pretreatment of human T-lymphocytes with a low dose of X-rays on the induction of mutations at the hprt locus by a subsequent challenge dose. When cells were exposed to 1 cGy X-rays 24 h after phytohemagglutinin stimulation, the yield of mutations induced by a 300 cGy X-ray dose given 16 h later was reduced by approximately 70% from the control level of X-ray-induced mutations. This indicates that this previously described adaptive response to low dose X-rays also results in lymphocytes becoming refractory to the induction of gene mutations.     

Rhodium(III) as a potentiator of the effects of X-rays on cells

A rhodium compound, Rh(NH3)3Cl3, does not sensitize the spores of Bacillus megaterium to X-rays. However, it is a very effective sensitizer of vegetative cells of Staphylococcus aureus, raising the sensitivity four times in O2 and over 100 times in anoxia. The inhibition by oxygen of the sensitizing action of Rh(III), which operates over a wide range of [O2], is noteworthy. These experiments were performed in saline-phosphate buffer using 50 kVp X-rays. The results are discussed in terms of the known radiation chemistry of this compound.     

Expression of Met-(-1) angiogenin in Escherichia coli: conversion to the authentic less than Glu-1 protein

A method for obtaining authentic human angiogenin utilizing an Escherichia coli recombinant expression system is described. A synthetic gene encoding angiogenin was placed into a vector for direct expression under the control of a modified E. coli trp promoter. The protein was produced by the bacteria in an insoluble form and purified to homogeneity by cation-exchange and reversed-phase HPLC following reduction/solubilization and reoxidation. The protein isolated was identified as Met-(-1) angiogenin by amino acid analysis and tryptic peptide mapping; the latter demonstrated that all three disulfide bonds had formed correctly. Both the enzymatic and angiogenic activities of the Met-(-1) protein were equivalent to those of native angiogenin. A Met-(-1) Leu-30 derivative of angiogenin was also isolated and found to be fully active. Conversion of Met-(-1) angiogenin to the authentic less than Glu-1 protein was achieved by treatment with Aeromonas aminopeptidase under conditions in which the new N-terminal glutamine readily cyclizes nonenzymatically. This aminopeptidase treatment may have more general applicability for removal of undesirable N-terminal methionine residues from foreign proteins expressed in bacteria.     

Enhanced ELISA: how to measure less than 10 picograms of a specific protein (immunoglobulin) in less than 8 hours

In this paper we outline a flexible and rapid method to measure picogram quantities of isotype-specific immunoglobulin (Ig), including IgE. Only readily or commercially available reagents are required: isotype-specific, anti-human Ig murine monoclonal antibodies (Mab) to coat microtiter plates, polyclonal alkaline phosphatase-coupled isotype-specific F(ab)'2 or Fab' fragments as second antibodies, and an enhanced developing system that amplifies the signal-to-noise ratio of the quantitatively bound second antibody. The procedure is detailed in the appendix to enable easy application, even if one has no previous experience with ELISAs. This system can be used to detect less than 10 picograms of Ig in cultures supernatants of cells that contain mixtures of various Igs and it can be used to detect the product of a single cell producing Ig. This method also will be applicable to measurement of the minute quantities of lymphokines and other biologically active molecules produced in vitro and found in various fluids in vivo.     

Melting curves on less than 1 μg of nucleic acid

Two types of microcuvettes were developed for the registration of melting curves in normal uv-spectrophotometers. Nucleic acid (0.5–1 μg) is required to determine changes in absorption with an accuracy of 0.1 mA. The quartz cuvette allows the registration of equilibrium melting curves up to 100°C. The other type of cuvette made from gilded brass allows to measure slow temperature jumps up to 80°C and equilibrium melting curves up to 70°C. The time range of ～3 s to several hours is accessible complementing the Eigen/De Mayer temperature-jump technique (1963, in Techniques of Organic Chemistry, Vol. VIII/2, pp. 895–1054, Wiley, New York). Temperature jumps of high amplitude as well as jumps to lower temperatures are possible. Examples of measurements on viroids and double-stranded viral RNA are presented.     

The effects of X-rays on the chromosomes of locust embryos

The variation in yield and the dose-response for chromatid aberration types following x-irradiation of Schistocerca gregaria embryo cells is described. Marked variations in yield are found for all aberration types during the G₂ and latter part of S stages of interphase. Only gaps appear to follow similar curves, other aberration types having unique patterns of response. The dose exponents for the various chromatid aberration types are similar to, but lower than, those reported for other organisms. Chromatid “breaks” appear to have a dose exponent greater than 1.0 — a fact which is in conformation with the exchange hypothesis. The chromosome radiosensitivity of this organism is similar to that reported for other organisms.     

The effects of X-rays on the chromosomes of locust embryos

Irradiation of embryo cells of Schistocerca gregaria in the two hours before mitotic anaphase produces a characteristic series of responses. An apparently insensitive stage is followed by the production of stickiness and side-arm bridges and finally by mitotic delay. Mitotic delay is complete at doses as low as 25 R. It is argued that stickiness is due to a generalised physiological process and that side-arm bridges are due to a localised exchange of discrete sub-units of the chromatid. These conclusions are based upon the kinetics of appearance and dose-response and a comparison of haploid and diploid cells in a haplo-diploid mosaic embryo.     

The effects of X-rays on the chromosomes of locust embryos

The structure and frequency of chromatid interchange types in C-metaphase cells of embryos of Schistocerca gregaria are described at the first mitosis following irradiation at the G ₂ stage of interphase. The relationship between the different interchange types and the organisation of the interphase nucleus is discussed. It is concluded that most chromatid interchanges are induced between polarised chromosomes though the occurrence of loops and to a lesser extent of terminal overlapping may subsequently modify the appearance of these aberrations at metaphase.     

The effects of X-rays on the chromosomes of locust embryos

Irradiation of Schistocerca gregaria embryo cells during the S and G₂ stages of interphase produces the same general classes of chromatid aberration as have been seen in organisms, such as Vicia faba, which have been used widely in radiocytological work. In addition, however, a series of intrachange aberrations has been found which is novel. In order to account for these Revell's exchange hypothesis has been extended to include the involvement of isolocus primary lesions. The exchange-type hypothesis is preferred because it can more readily accomodate the whole spectrum of aberrations found.     

Periodic analysis of ultradian (40 min less than tau less than 24h) respiratory variations in laboratory vertebrates of various circadian activities

Continuous recordings of respiratory gas exchanges of various laboratory endotherm vertebrate species, which have either a nocturnal (mouse, rat) or diurnal (monkey, quail, chicken) or equivocal (guinea-pig) maximal activity, kept under controlled environmental conditions of temperature, humidity, ventilation and provided with food and water ad libitum, show ultradian oscillations of mean and low frequencies (1 less than f less than 35 c.day-1). Harmonic analysis was used to assess periodic or random ultradian variations and to compute amplitudes and phases of these oscillations when these vertebrates were submitted to a light (100 lx) and dark circadian alteration (LD 12:12). Spectral analysis shows that a 100-lx continuous illumination or continuous darkness decreases circadian respiratory rhythms and increases these ultradian respiratory oscillations.     

A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins

We have developed conditions for the efficient electrotransfer from polyacrylamide gels to nitrocellulose sheets of a broad size range of proteins (Mr 8,000 to Mr greater than 400,000). The important features of this procedure include a two-step electrotransfer, beginning with elution of low-molecular-weight polypeptides at a low current density (approximately 1 mA/cm2) for 1 h, followed by prolonged electrotransfer (16-20 h) at high current density (approximately 3.5-7.5 mA/cm2) in conditions that favor the elution of high-molecular-weight proteins. The transfer buffer includes 0.01% sodium dodecyl sulfate to enhance protein elution, and 20% methanol to improve the retention of proteins on the nitrocellulose sheet. The nitrocellulose is air-dried after transfer is complete to eliminate loss of proteins during subsequent processing. This transfer procedure works well with proteins prepared from many different cell types, and is suitable for use with all polyacrylamide gel systems tested. With little or no modification, our method should also be applicable to transfer membranes other than nitrocellulose.